The detection of trace amounts of biologically significant compounds, such as steroids, drugs, selected antigens, or tumor markers, is often accomplished inexpensively by the employment of an immunoassay. Enzyme immunoassay (EIA) methods are common antigen detection techniques. One of the most common types of immunoassays is the Enzyme-Linked Immunosorbant Assay (ELISA), a solid phase enzyme immunoassay technique. Such assays rely on an immunogenic recognition of a substance in question followed by the amplification of the signal generated by that recognition. Enzymes are widely used in immunoassays as the amplifier of the antibody-antigen recognition event. Traditionally, antigen capture assays involve the application of an antigen-containing sample to a plastic plate where a “capture” antibody has been previously bound. A secondary (“detection”) antibody is then applied and binds to the antigen. This binding forms a sandwich that leads to the quantification of antigen present. EIAs are easy to multiplex and the use of more than one antibody in the sandwich assay improves the specificity of the test by requiring two specific interactions before signal is detected.
ELISA may be preformed in a number of different ways. The two most common are the competitive mode and the sandwich assay. In a competitive mode ELISA, a surface, usually either a polystyrene plate or a nitrocellulose membrane, is coated with a capture antigen. These surfaces are normally chosen because they bind protein non-specifically. Therefore, if the antigen is not a protein, it may be covalently linked to a carrier protein and bound to the surface without further chemistry. After the antigen is bound, the remaining binding sites on the surface are blocked with another protein as a blocking agent. Then the test fluid and enzyme-labeled antibody are added. If no antigen is in the test fluid, the labeled antibody will bind to the antigen adsorbed on the surface. Conversely, if antigen is present in the test fluid, the antigen will block the binding sites on the enzyme-labeled antibody and prevent it from binding to the antigen adsorbed on the surface. The surface is washed to remove unbound materials, and a substrate is added for the enzyme. The enzyme catalyzes a reaction in which the substrate reacts to form a colored material that can be quantitatively measured with a spectrophotometer. The intensity of the color produced is proportional to the enzyme activity and the amount of antibody bound, which is inversely proportional to the amount of antigen in the test fluid.
In a sandwich assay ELISA, an antibody that recognizes part of the antigen is bound to a surface. Since antibodies are proteins, this is readily accomplished by allowing the surface to contact a solution of the antibody. As in the competitive ELISA, the remaining sites on the surface are blocked with another protein as a blocking agent. The test fluid is then added. If an antigen is present in the test fluid, the antibody on the surface will capture the antigen. Then a second, enzyme-labeled antibody, which recognizes a different part of the antigen than the first antibody, is added. The second antibody will then bind to the antigen that is captured on the surface. After washing the surface to remove any unbound materials, a substrate for the enzyme is added and the color produced is measured spectrophotometrically. In this form of an ELISA, the signal is directly proportional to the concentration of the antigen in a test sample. Such a sandwich assay is widely used in the commercial arena, e.g., for home pregnancy tests.
In either type of ELISA, the enzyme acts as the amplifier of the antigen-antibody reaction. That is, a color or other signal, such as light from some chemiluminescent reaction, is produced that can be observed macroscopically. Without this amplification step, the sensitivity of an immunoassay would be orders of magnitude less.
Several problems occur in the use of enzymes as amplifiers in immunoassays including:
1) Any change in enzyme activity will affect the precision of the assay. For example, loss of half of the activity of the enzyme in a competitive ELISA may produce a false positive since a smaller signal indicates the presence of the test substance. Since enzyme activity is sensitive to storage conditions, enzymes must be kept either refrigerated, freeze-dried or both. Also, controls must be performed to constantly test the activity of the enzyme. Inevitably, the shelf-life is limited by the stability of the enzyme.
2) Enzymes are expensive as they are derived from living sources and require substantial processing costs. The least expensive enzyme, on an activity basis, is Horseradish Peroxidase, which is, not surprisingly, the most common enzyme used in ELISAs. However, even Horseradish Peroxidase costs about $5/mg or $5000/g. Fortunately, very little enzyme is necessary for each assay.
3) The labeling of antibodies with enzymes is often a quite laborious procedure, as one must ensure that little unbound enzyme is present. If significant amounts of unbound enzyme are present or significant amounts of unlabeled antibody are present, the sensitivity of the ELISA is reduced.
4) Enzymes are often heterogeneous materials due to their isolation from natural sources. Therefore, characterization of enzyme-antibody conjugates can be difficult.
Although EIAs performed on 96-well plates are popular in the academic lab, modem clinical labs employ more highly automated assay systems. One example is the Abbott disposable IMx® cartridge system, which utilizes fluorescent polarization. In this approach, a capture antibody is bound to a microparticle, and the sample is incubated with the filter and a fluorescently labeled detection antibody. Since the unbound detection antibody has no net fluorescent polarization compared to the bound detection antibody, the fluorescent polarization signal is proportional to the amount of bound sample. Although this assay system does not employ an enzymatic amplification step, it is still very sensitive, and it has other advantages, including the elimination of time consuming wash steps.
Other automated systems involve other approaches to save time, such as using kinetic rather than equilibrium approaches to measure product. Other detection systems include exotic methods such as electrochemiluminescence (ECL), where the capture antibody is bound to magnetic beads and the detection antibody is labeled with a Ru(bipyridyl)3 complex. After incubation and washing, the ruthenium complex emits light in an electrochemical cell. This assay system can detect antigens in the low picomolar (pM) range. All the above assay systems are performed in clinical labs on expensive equipment and are not available as physician operated desktop systems with untrained professionals.